In this video-article we present a step-by-step demonstration on how to collect and cryopreserve hamster oocytes with high post-thaw survival rates. The same procedure can also be applied to successfully freeze and thaw mouse embryos at different stages of preimplantation development.
Animal care and procedures were conducted according to the guidelines and regulations approved by the Ethics Committee on Animal and Human Research of the Universitat Autònoma of Barcelona, Spain.
I. Oocyte collection
For the collection of mouse embryos, female mice (Mus musculus; 6-8 week’s old) are induced to superovulate by intraperitoneal injection of 5 IU of PMSG followed by 5 IU of hCG 48 hours later and mated with male mice. Females are sacrificed by cervical dislocation at 24-26, 44-46 or 54-56 hours after the administration of hCG for obtention of pronuclear, 2-cell or 4-cell stage embryos, respectively. Collection of pronuclear embryos is performed exactly as described for hamster oocytes. Embryos at the 2-cell and 4-cell stages are collected by flushing the oviducts with KSOM-H medium [6].
Note: Following this protocol approximately 20 to 30 oocytes/embryos can be collected from each female.
II. Preparation of freezing and thawing solutions
Note: Solution I and freezing and thawing solutions must be prepared just before their use. However, the specified amounts of sucrose to be used for the preparation of the freezing and thawing solutions may be weighted in advance and stored in 3 ml-tubes at room temperature for several months.
III. Freezing protocol
IV. Thawing protocol
Note: During steps 6 and 7 you should see that the thawed oocytes/embryos slowly swell up due to rehydration.
V. Representative Results
The use of the cryopreservation protocol reported here results in a survival rate of approximately 95% for hamster oocytes and of >90%, 85% and 80% for mouse B6CBAF1 embryos at the pronuclear, 2-cell and 4-cell stages, respectively. When mouse embryos are cultured in vitro in KSOM after thawing, at least 70 to 80% should reach the blastocyst stage in optimal culture conditions.
It is recommended that you include a control group (straw) of oocytes/embryos in each freezing experiment and that you thaw this group before the rest of the oocytes/embryos are used for further manipulations. If survival rates for the control group are lower than expected, thaw another straw from the same lot. If survival rates for this second group are also low, discard the remaining straws from this freezing lot.
With this protocol hamster oocytes and mouse embryos can be successfully cryopreserved. Once frozen, oocytes/embryos can be stored in liquid nitrogen tanks indefinitely and recovered at any time or place desired. This offers the advantage of having samples almost ready to use whenever needed. On the other hand, this protocol can also be used to generate embryo cryobanks from valuable mouse strains (such as transgenic lines), as an economical and safer alternative to the maintenance of live animal colonies. It is important to note that this protocol is optimized to cryopreserve hamster oocytes and hybrid B6CBAF1 mouse embryos. Differences in post-thaw survival rates among mouse embryos with different genetic backgrounds have been described 8,9, thus care should be taken to assess cryopreservation conditions on each species or strain of oocytes/embryos and to establish the appropriate protocol before the generation of oocyte or embryo cryobanks.
Hamster oocytes can be used in human assisted reproduction clinics for the sperm penetration test or for sperm chromosome analysis 10, or as practicing material for beginners in intracytoplasmic sperm injection (ICSI). On the other hand, mouse hybrid B6CBAF1 embryos at pronuclear or 2-cell stages can be used in human assisted reproduction centers or in research labs to check the quality of the embryo culture media or the proper functioning of the incubators.
This work was supported by the Spanish Ministerio de Educación y Ciencia (BIO 2006-11792) and the Generalitat de Catalunya (2005-SGR00437). Authors would like to thank Marc Puigcerver and Jonatan Lucas for their technical assistance in preparing the media. All staff from the Servei d’Estabulari is acknowledged for animal housing, and especially Juan Jose Martin and Javier Benito for their additional contribution on recording part of this video. N.C.B. is a fellow of the Portuguese Fundação para a Ciência e Tecnologia and S.G. is a fellow of the Spanish Ministerio de Educación y Ciencia.
Material Name | Type | Company | Catalogue Number | Comment |
---|---|---|---|---|
French type mini-straw 0.25 ml | Mini-tub | 13407/0010 | ||
hCG | Farma-Lepori, Spain | 749036.4 | ||
Hyaluronidase | Sigma | H-3884 | ||
KSOM-H medium | Ref. [11] | |||
Liquid Nitrogen | Air Liquide | |||
Plastic plugs | Mini-tub | 19046 | ||
PMSG | Intervet, Spain | 1.614/8447 | ||
Propilenglycol | Fluka | 82280 | ||
Sucrose | Merck | 107687 | ||
Surgical Scissors | Aesculap | BC-060R; BG-061R | ||
Thermo-sealer | SIZ | 220 | ||
35 and 90 mm culture dishes | Nunc | 153066; 150350 | ||
10 ml tubes | Greiner Bio-one | 163160 | ||
Nitrogen tank | MVE, Cryogenics | XC 43/28 | ||
Programmable Freezer | Planner Products Ltd. | Kryo-10 Series III | ||
Forceps | Aesculap | BD-331R; BD-053R; OC-020R |